Intracellular Calcium (Ca) is a key cellular messenger that triggers a variety of cellular processes. This messenger Ca can come from internal stores, or can enter the cytosol from the extracellular medium. Ca can be released from intracellular stores via at least two types of Ca channel: the inositol 1,4,5-trisphosphate receptor (InsP3R) and the ryanodine receptor (RyR). This proposal addresses the integrative regulation of Ca release via the IsnP3R using biophysical, biochemical, and immunological techniques. We will begin these studies of cellular regulation using the richest known source of IsnP3R, the cerebellum. These brain channels will provide a model for subsequent studies of the regulation of less abundant smooth muscle and cardiac InsP3R. Specific long-term goals of this project are to understand how the InsP3-gated channel functions, how the cell regulates the channel to optimize cellular responses, and how regulation goes awry in pathophysiological situations. In the proposed experiments, we will monitor the activity of native and purified InsP3-gated channels after incorporation into planar lipid bilayers. In the first series of experiments in this proposal, the InsP3R will be studied using biophysical techniques to determine how the receptors are regulated, in particular by cytoplasmic Ca. In the second series of experiments, channel properties of the InsP3R will be studied with emphasis on an investigation into the role of luminal Ca in the Ca- dependent regulation of the channel. The third series of experiments will examine channel components necessary for Ca regulation of channel function by testing the role of associated proteins and utilizing antibodies targeted to portions of the InsP3R thought to be involved in regulation of receptor function by Ca. The many interacting levels of regulation of Ca release, which utilize several different intracellular Ca channels, provide for both positive feedback (amplification) and negative feedback (inhibition) of the intracellular Ca signal. Identification of the molecular mechanisms controlling these channels will assist in the understanding of processes as diverse as muscle contraction and motor learning, and may provide insights into conditions such as hypertension and heart failure, processes that involve abnormalities in the activity of these channels.